A1.20 Epigenetic modificationsand TLR8 signalling contribute to systemic sclerosis pathogenesis by ros and profibrotic genes induction

Background and objectivesSystemic sclerosis (SSc) is a chronic multisystem autoimmune disease characterised by skin and internal organs fibrosis and immune abnormalities. Recent evidence suggests that activated circulating monocytes from SSc patients play a role in SSc pathogenesis due to enhanced expression of tissue inhibitor of metalloproteinases 1 (TIMP-1), IL-8 and reactive oxygen species (ROS) induction, which contribute to fibrosis progression and chronic inflammation. The exact factors driving TIMP-1, IL-8 and ROS secretion are still unknown.The aim of this study was to investigate the expression pattern of profibrotic IL-8, TIMP-1, AP1 transcription factor-Fra2 and ROS induction in peripheral blood monocytes following DZNep (histone methyltransferase inhibitor) and TLR8 agonist (ssRNA) stimulation.Materials and methodsThe expression of Fra2, IL-8 and TIMP-1 and anti-oxidant superoxide dismutase 1 (SOD1) was measured by qRT-PCR in stimulated and unstimulated HC (n = 14) and SSc (n = 17) monocytes. Generation of ROS was determined using luciferase based assay. Fra2 DNA-binding activity was measured in AP-1 transcription assay in monocytic U937 cell line following epigenetic and TLR8 modifications. The level of anti-fibrotic miRNA-5196, which is predicted to bind and inhibit 3’UTR of Fra-2 gene, was also determined in HC and SSc monocytes.ResultsCombination of DZNep+TLR8 enhanced Fra2 (2-fold, p = 0.02), TIMP-1 (2-fold) and IL-8 (7.87-fold, p < 0.001) expression in SSc monocytes. Fra2 DNA-binding activity was 1.5-fold increased upon stimulation. Secreted level of TIMP-1 was 1.46-fold higher in SSc monocytes compared to unstimulated cells. Generated ROS was 2.21-fold (p = 0.0395) higher following DZNep+TLR8 stimulation in monocytic U937 cells. In contrast, miRNA-5196 expression was 2.13-fold decreased in SSc monocytes upon DZNep+TLR8 stimulation. Also the level of SOD1 was decreased in HC and SSc monocytes following stimulation, 2.16-fold (p = 0.025) and 1.56-fold, respectively.ConclusionsThese data suggest that DZNep and TLR8 agonist are able to enhance pro-fibrotic TIMP-1, IL-8 and oxidative stress generation. As opposed by the decrease of anti-fibrotic miRNA-5196 and anti-oxidant SOD1 expression in SSc monocytes, which might be used as a potential modulators of fibrogenesis in SSc.Supported by Homing Plus grant/2013–8/4 from Foundation for Polish Science and UMO-2015/16/S/NZ6/00041 from National Science Centre, Poland.